CN101389760A - Peptides effective in the treatment of tumors and other peptides requiring the removal or destruction of cells - Google Patents

Abstract

The invention includes methods of treating conditions requiring removal or destruction of cellular elements, such as benign or malignant tumors in humans, using compounds based on small peptides. The method includes, but is not limited to, administering the compounds intramuscularly, orally, intravenously, intrathecally, intratumorally, intranasally, topically, transdermally, etc., either alone or conjugated to a carrier.

Description

The peptide of the illness of cell need be removed or destroy to effective treatment tumour or other

The cross reference of related application

The application requires the U.S. Provisional Application No.60/776 of submission on February 28th, 2006, and 933 right of priority is incorporated them for your guidance in full at this.

Background of invention

1. invention field

The present invention includes the compounds for treating of using based on little peptide and need remove or destroy cell element (cellularelement), as the method for the human disorders of optimum or malignant tumour.Described method includes, but not limited in the intramuscular, per os, intravenously, sheath, in the tumour, in the nose, part, transdermal etc. are independent or unite with carrier and to give described compound.

2. description of related art

The essence of many therapeutic treatments and treatment process comprises removes or destroys harmful or unwanted tissue.The example of this critical treatment comprises excision cancerous growths thing, destroys metastatic tumo(u)r and reduce gland (as prostate gland) hyperplasia by chemotherapy.Other example comprises to be removed unwanted facial hair, remove wart and removes unwanted fatty tissue.

The significant need potent agent can destroy so that remove or suppress harmful or do not need the further growth of cell and tissue, but mainly has local effect and general toxicity is minimum or do not have.

Such reagent is described in waits to examine U.S. Patent Application Serial Number 10/092,934, be entitled as " with the method (Methods of Treating Tumors and RelatedConditions Using Neural Thread Proteins) of using neural thread protein treatment tumour and associated conditions "; Sequence number 10/153,334, be entitled as " effectively treat tumour and need remove or destroy the peptide (Peptides Effective In The TreatmentOfTumors And Other Conditions Requiring The Removal Or Destruction OfCells) of the illness of cell " with other; Sequence number 10/198,069, be entitled as " effectively treat tumour and need remove or destroy the peptide (Peptides Effective In The Treatment Of Tumors And Other ConditionsRequiring The Removal Or Destruction Of Cells) of the illness of cell " with other; Sequence number 10/198,070, be entitled as " effectively treat tumour and need remove or destroy the peptide (Peptides Effective In TheTreatment Of Tumors And Other Conditions Requiring The Removal OrDestruction Of Cells) of the illness of cell " with other; Sequence number 10/294,891, be entitled as " effectively treat tumour and need remove or destroy the peptide (Peptides Effective In The Treatment Of Tumors AndOther Conditions Requiring The Removal Or Destruction Of Cells) of the illness of cell " with other; And sequence number 10/920,313, be entitled as " effectively treat tumour and need remove or destroy the peptide (Peptides Effective In The Treatment Of Tumors And Other Conditions RequiringThe Removal Or Destruction OfCells) of the illness of cell with other ", it is for reference that their contents are separately all included this paper in.

What this paper disclosed is composition, fragment and the subsequence of a kind of this type of peptide reagent (SEQIDNO.1:Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Gl u-Ile-Lys-Arg-Cys-Leu), and they also can be used for treating tumour and other need remove or destroy the illness of cell.

Cancer is the unusual of cell interior regulation mechanism generation, causes uncontrolled cell growth and breeding.Normal cell constitutes tissue, and when these cells lost as specialized, controlled and collaborative unitary ability (dedifferenting), it is chaotic that this defective can cause cell mass to take place.When it takes place, promptly form tumour.

The optimum hypertrophy of tissue is abnormal conditions, and need remove cell this moment from body.Innocent tumour is that it( ) can whole body do not shift but can cause the cell proliferation of condition symptoms.If this tumour is arranged in the zone that organ such as brain are difficult to arrive, they may be fatal.Innocent tumour is present in many organs, comprises lung, brain, skin, hypophysis, Tiroidina, adrenal cortex and adrenal medulla, ovary, uterus, testis, reticular tissue, muscle, intestines, ear, nose, larynx, tonsil, mouth, liver, gall-bladder, pancreas, prostate gland, the heart and other organs.

The normally the first step of in cancer therapy, performing the operation.The purpose difference of operation.Sometimes remove tangible tumour as much as possible with operation, or make it " remove piece " (reducing the needs that other method is treated) at least thereby remove main tumor mass.Depend on cancer kind and position, operation also can be patient provides some remissions.For example, if excision the cerebral tumor of most of amplification, intracranial pressure will reduce, and make patient's doing well,improving.

Not every tumour all is suitable for operation.Some tumours may be positioned at the body part that can not remove fully.These tumour examples are the tumours in the brain stem (part of control breathing in the brain) or are grown in main blood vessel neutralization tumour on every side.In these situations, because the relevant excessive risk of tumor resection, the effect of operation is limited.

In some cases, without the ocal resection piece, because simply do not need.An example is a Hodgkin lymphoma, and this is the lymphoglandula cancer that combination has sound response to chemotherapy and radiation.In Hodgkin lymphoma, seldom needing performs the operation realizes curing, and operation nearly all is to be used for determining diagnosis.

Chemotherapy is the another kind of common type of cancer therapy.It comprises that in fact use can specificity attacks in the whole body medicine (giving by oral or injection usually) of somatoblast fast (as the cell seen in the tumour).It is high but obviously do not surpass the tumour at primary tumor position as yet that this makes chemotherapy can be used for treating metastatic carcinoma and the possibility by blood and lymphsystem diffusion.Chemotherapy also can be used for improving the reaction of local tumor to operation and radiotherapy.For example for the cancer of some heads and neck.

Unfortunately, normal other cell of splitted (as the endodermis and the hair of stomach) fast also can be subjected to the chemotherapy influence in the human body.For this reason, many chemotherapeutics can cause adverse side effect as nauseating, vomiting, anaemia, alopecia or other symptom.These side effects are temporary, and present medicine can help to alleviate many these side effects.Along with our knowledge sustainable growth, scholars have designed the chemotherapeutics that upgrades, and they are kill cancer cell and littler to patient's side effect better.

Chemotherapeutics can give patient in many ways.Some are pills, and some are by intravenously or other position drug administration by injection.With regard to injectable chemotherapeutics, patient must receive treatment to Doctor's office or hospital.Other chemotherapeutics needed import in the blood flow continuously in one day 24 hours.For the chemotherapeutics of these types, can carry out minor operation and implant little pump to patient.With slowly administration of pump.In many cases, must in venous patient, settle permanent tubing port in order to avoid need repeated puncture.

Radiotherapy is the another kind of anticancer weapon of using always.Radioactive rays kill cancer (cell) by the DNA that destroys in the tumour cell.Ray can transmit with different modes.Prevailing method comprises in the high precision mode makes radiation beams point to patient, and cover is on tumour.For this reason, patient must lie on the platform light beam and moves around him.But this Cheng Chixu several minutes, but but carry out every day, several weeks (depending on tumor type) are to reach specific total prescription exit dose continuously.

Sometimes adopt the another kind radiation method that is called brachytherapy, comprise and adopt radioactivity piller (" radiation seed (seed) ") or electric wire, they are implanted in the tumor region of health.Implant can be temporary transient or nonvolatil.With regard to permanent implanted, the ray in the radiation seed in several days or a few week " decay " thus patient does not have radioactivity.With regard to temporary implantation, total radiation dose must be imported in a couple of days usually, patient at this moment between in must be in hospital.For these two kinds of brachytherapies, generally radioactive rays must be sent precise region to obtain part control (this and whole body therapeutic, opposite) as chemotherapy to cancer.

Some patients that highly select go to bone marrow transplantation for help.Usually carry out this kind treatment and be because patient suffers from significant invasive carcinoma or because their cancer return behind conventional therapy for treating.Bone marrow transplantation is the process of a complexity.There are numerous species and them to cause that the possibility of side effect and healing is different.Most of transplanting must be carried out in specific medical centre, adopt to transplant in many cases and to think the property studied.

Many other methods of treatment are arranged,, do not become standard treatments in clinical examination although major part is still explored.Example comprises immunotherapy, monoclonal antibody, anti-angiogenesis and gene therapy.

Immunotherapy: designed multiple technologies to promote patient's autoimmunization system opposing cancer, these are different fully with radiotherapy or chemotherapy.For reaching this purpose, scholars' special vaccine commonly used is given patient injection.

Monoclonal antibody: utilize the difference of antigenicity between cancer and the non-cancer cells or/or further feature to be designed for the antibody that adheres to cancer cells (but not normal cell).Antibody can give patient separately, or with various cytotoxic compound couplings or adopt the radioactivity form, thereby make the preferential target cancerous cells of antibody, by this toxic agents or radiological agent are delivered to the cell that needs.

Anti-angiogenesis: because cancer cells divides rapidly and tumor growth, they may be looked very soon above its blood supply.For compensating this point, some tumours can be secreted and be it is believed that the material that can assist to induce its surrounding blood vessel growth, thereby the nutrition in blood vessel source is provided to cancer cells.Designed experimental therapy to stop the blood vessel tumour of growing into.

Gene therapy: cancer is the product of a series of sudden changes, finally causes cancer cells to produce and hyper-proliferative.The prodrug that procedural cell (death) mechanism that the treatment cancer can introduce cancer cells by the gene that will stop or stop cancer propagation, open cell can be transformed into toxic metabolites with destruction of cancer cells, the immunity identification that strengthens cell or expression or the cytokine of inhibition tumor growth.

Innocent tumour and deformity also can be treated by several different methods, comprise operation, radiotherapy, pharmacological agent, heat and galvanocautery, psychrotherapy etc.Although innocent tumour does not shift, they can be grown up and can recur.The surgical eradication innocent tumour is had any problem and surgery side effect usually, and some innocent tumours need undergo surgery repeatedly as pituitary adenoma, meningioma, hyperplasia of prostate etc.

Also have some other illness to comprise the unwanted cells element, need these cells of selective removal.For example heart disease and stroke is generally caused by atherosclerosis, and atherosclerosis to be the proliferative damage of the unstriated muscle element of fiber fat and modification cause the vessel wall distortion, lumen of vessels is narrow, blood flow shrinks, susceptible disease kitchen range thrombus and finally cause blocking and blocking.Treating atherosclerotic the whole bag of tricks comprises: bypass graft; The artificial graft; Adopt revascularization, strike off, radiation, laser or other remove the angioplasty of blocking; Suppress atherosclerotic pharmacotherapy by reducing lipid; The anticoagulant treatment; Conventional means with diet, exercise and mode of life.Still need a kind ofly to remove atherosclerotic lesions and do not have the risk of surgical procedure and the method for side effect.

Other needs the example of the undesired cell element of selective removal to comprise the grower such as the wart of virus induction.Another example is loose inflammatory piece and loose scar or the keloid seen in the inflammation situation.For example remove unwanted hair such as facial fine hair when other example sees make-up and beauty, or must remove in skin of face and the reticular tissue or the unwanted tissue regions of shrinkage in four limbs skin and the reticular tissue for cosmetic purpose.

Other need selecting cell to remove or the example that does not need the cell element that suppresses cell proliferation comprises any artery, valve or pipe in the recycle system, include but not limited to valve (for example comprising that the narrow Aorta of aortic valve fenestra is narrow), (for example comprising crown mouthful of sclerosis of stenosis of coronary orifice) coronarius, carotid artery and arteriorenal narrow or restenosis.Other examples comprise and suppressing or growth of removal undesirable cell or gathering, these growths or gathering cause for example placing or transplant in intravascular in treatment narrow, constriction or aneurysma wherein, or the medical facilities that place or transplant in urethra and bile duct partially or completely block.

Other example is that this field those skilled in the art know.In all or most of these examples, all need to remove or to destroy undesired cell element and risk and side effect and the more accurate methods of treatment of not wanting the cell element of removing of not having conventional therapy.

In comprising the entire description of above-mentioned association area in being described in, any and all open source literatures as herein described comprise any and all United States Patent (USP)s are all special includes this paper in for your guidance in full.The description of above-mentioned association area is not to admit by any way to comprise that the document any described herein of waiting to examine U.S. Patent application is a prior art of the present invention.In addition, described herein and these products, method and/and the relevant shortcoming of instrument are not in order to limit the present invention.In fact, each side of the present invention can comprise some feature of described product, method and/or instrument, and not limit by its shortcoming.

Summary of the invention

This field still needs the method for treatment unwanted cells element new, that toxicity is more weak.The present invention can satisfy these demands.

The present invention's part comprises the particular peptide that aminoacid sequence Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu describes based on finding some peptide, can treat and/or kill unwanted cells propagation.These unwanted cells propagation comprise for example optimum and malignant tumour, gland (as prostate gland) hyperplasia, unwanted facial hair, wart and unwanted fatty tissue etc.

The present invention's part is based on this amazing and unexpected discovery, and promptly unwanted cells propagation also can be treated and/or kill to some peptide fragment of peptide Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-GIu-Ile-Lys-Arg-Cys-Leu (" S05A peptide ") and subsequence.

Some embodiments relate to the method for treatment unwanted cells propagation (optimum or malignant tumour, gland (as prostate gland) hyperplasia, unwanted facial hair, wart and unwanted fatty tissue), and this method comprises the S05A peptide of the Mammals treatment significant quantity that these needs are arranged.

This S05A peptide can be used separately or in conjunction with carrier or antibody.With by in (essence in), Intraventricular, the tumour in intramuscular, per os, intravenously, intraperitoneal, the brain, in the intralesional (interlesionally), intradermal, sheath, in the nose, intraocular, intra-arterial, part, transdermal, by aerosol, inculcate, inject, transplantation device, slow-released system etc. are separately or in conjunction with vector administration S05A peptide.In addition, because hereditary change or alternate manner, can be by using the gene of expressing the S05A peptide, inducing vaccine that this class generates or express the S05A peptide in vivo by cell, bacterium or the virus of introducing this peptide of expression in vivo by using.

In addition, the S05A peptide can be treated agent in conjunction with other and makes and be used for treating optimum and malignant tumour and other does not need or the growth of deleterious cell.

Above-mentioned general description and following detailed description are exemplary and illustrative, be to declare to invent further specify.From following detailed Description Of The Invention, other target, advantage and feature are conspicuous for those skilled in the art.

Preferred implementation describes in detail

Before describing protein of the present invention, nucleotide sequence, peptide etc. and method, should understand and the invention is not restricted to described concrete grammar, scheme, clone, carrier and reagent, because these can change.Should also be appreciated that term used herein only for describing specific embodiment, is not to limit the scope of the invention, this scope is only limited by claim.

Except as otherwise noted, term used herein and phrase by as give a definition.

Unless spell out in addition in the literary composition, in entire description, odd number " " comprises plural meaning.Therefore for example " host cell " comprises a plurality of such host cells, addressed one or more antibody and Equivalent well known by persons skilled in the art thereof and address " a kind of antibody ", and the rest may be inferred.

Can address amino acid as herein described and amino-acid residue according to single-letter of being accepted or three-letter code that following table provides.

Table 1

Three alphabetical amino acid	One-letter symbol	Symbol
			L-Ala	A	Ala
Arginine	R	Arg
			L-asparagine	N	Asn
Aspartic acid	D	Asp
			Halfcystine	C	Cys
Glutamine	Q	Gln
			L-glutamic acid	E	Glu
Glycine	G	Gly
			Histidine	H	His
Isoleucine	I	Ile
			Leucine	L	Leu
Methionin	K	Lys
			Methionine(Met)	M	Met
Phenylalanine	F	Phe
			Proline(Pro)	P	Pro
Serine	S	Ser
			Threonine	T	Thr
Tryptophane	W	Trp
			Tyrosine	Y	Tyr
Xie Ansuan	V	Val

Term " peptide " in this article refers to has two amino acid whose chains at least, comprises homologue, derivative, fragment and the variant of this peptide.Statement " S05A peptide " refers to contain at least one fragment of peptide shown in the SEQ ID NO.1 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu) or the peptide of subsequence, except as otherwise noted, any homologue, fragment, derivative, variant, fusion rotein and the peptide mimics that also comprise this peptide.Statement " S05A peptide " includes, but is not limited to comprise the peptide that at least one is selected from down the peptide of group:

A) peptide represented of the aminoacid sequence among the SEQ ID NO.2 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile);

B) peptide represented of the aminoacid sequence among the SEQ ID NO.3 (Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu);

C) peptide represented of the aminoacid sequence among the SEQ ID NO.4 (Val-Leu-Ser-Arg-Ile-Lys);

D) peptide represented of the aminoacid sequence among the SEQ ID NO.5 (Arg-Ile-Lys-Leu-Glu-Ile-Lys);

E) peptide represented of the aminoacid sequence among the SEQ ID NO.6 (Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu); With

F) peptide represented of the aminoacid sequence among the SEQ ID NO.7 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile).

Term " fragment " or " subsequence " are meant protein or the polypeptide that the continuous subsequence by the aminoacid sequence of protein or peptide constitutes, and comprise the fragment of natural generation, as the fragment of splice variant and the natural generation of body endoprotease activity.This fragment can (as natural montage) brachymemma in N-terminal, C-terminal and/or centre.This produced in fragments can be become contain or do not contain the N-terminal methionine(Met).Term " fragment " comprises the fragment from same protein or peptide, can be identical or different, and the aminoacid sequence that has or do not adjoin directly or by joint connects together.Consequently, comprising the segmental any peptide of SEQ ID NO.1 can be to be selected from above any peptide, and other fragments or subsequence will be that those skilled in the art are conspicuous, for repeating no more in the text for simplicity.The technician utilizes guidance listed in the text and method can select suitable fragment to be used for the present invention.

Term " variant " refers to a kind of protein or polypeptide, wherein compares with the aminoacid sequence of this albumen or peptide to have one or more aminoacid replacement, disappearance and/or insertion, and comprises the albumen of natural generation or the allele variant or the alternative splicing variant of peptide.Term " variant " comprises with the one or more amino acid in similar or homologous amino acid or the dissimilar amino acid whose substituted peptide sequence.Can be divided into amino acid similar or homologous (Gunnar von Heijne, " molecular biological sequential analysis " (Sequence Analysisin Molecular Biology), 123-39 page or leaf according to many standards, academic press (Academic Press), New York, New York, 1987).Preferred variant comprises that the L-Ala of one or more amino acid positions replaces.Other preferably replaces and comprises that conservative property little to proteinic whole net charge, polarity or hydrophobicity influence or that do not have to influence replaces.Conservative property replaces and to be listed in the table below in 2.

Table 2

Conservative amino acid replaces

Alkalescence:	Arginine Methionin Histidine
		Acid:	The L-glutamic acid aspartic acid
Not charged polarity:	Glutamine l-asparagine serine threonine tyrosine
		Nonpolar:	Phenylalanine tryptophane halfcystine glycine L-Ala Xie Ansuan proline(Pro) methionine(Met) leucine Isoleucine

Table 3 is listed the another kind of scheme of aminoacid replacement:

Table 3

Original residue	Replace
		L-Ala	Glycine; Serine
Arginine	Methionin
		L-asparagine	Glutamine; Histidine
Aspartic acid	L-glutamic acid
		Halfcystine	Serine
Glutamine	L-asparagine
		L-glutamic acid	Aspartic acid
Glycine	L-Ala; Proline(Pro)
		Histidine	L-asparagine; Glutamine
Isoleucine	Leucine; Xie Ansuan
		Leucine	Isoleucine; Xie Ansuan
Methionin	Arginine; Glutamine; L-glutamic acid
		Methionine(Met)	Leucine; Tyrosine; Isoleucine
Phenylalanine	Methionine(Met); Leucine; Tyrosine
		Serine	Threonine
Threonine	Serine
		Tryptophane	Tyrosine
Tyrosine	Tryptophane; Phenylalanine
		Xie Ansuan	Isoleucine; Leucine

Other variant can be made up of the less aminoacid replacement of conservative property, keep the structure that (a) replaces polypeptide main chain in the zone as the residue of selecting at them, for example lamella or helicoidal configuration, (b) electric charge of molecule target site or hydrophobicity, (c) the effect difference of side chain size is more remarkable.Usually function is had the replacement of more remarkable effect is that (a) glycine and/or proline(Pro) are by another kind of aminoacid replacement or disappearance or insertion in expection; (b) wetting ability residue such as seryl or threonyl replace (or quilt) hydrophobic residue, as leucyl, isoleucyl-, phenylalanyl, valyl or alanyl (replacement); (c) cysteine residues replaces (or quilt) any other residue (replacement); (d) have the residue of positive charge side chain such as residue such as glutamy or the aspartoyl (replacement) that lysyl, arginyl or histidyl-replacement (or quilt) have negative charge; Or the residue such as the phenylalanine that (e) have bulky side chain replace residue such as the glycine (replacement) that (or quilt) do not have this side chain.Other variant comprises and is designed to produce new glycosylation and/or phosphorylation site, or is designed to lack those residues of existing glycosylation and/or phosphorylation site.Variant comprises at least one aminoacid replacement at glycosylation site, proteolytic enzyme cutting site and/or cysteine residues place.Variant also is included in albumen or the peptide that has other amino-acid residue before or after this albumen on the joint peptide or the peptide ammino acid sequence.For example, thus cysteine residues can be added in the amino of S05A peptide and C-terminal makes this peptide cyclisation to form disulfide linkage.Term " variant " also comprises the polypeptide of the aminoacid sequence with S05A peptide, and it is connected at least 1 and to nearly 25 or how extra amino acid at 3 ' or 5 ' end side of S05A peptide.

Term " derivative " refers to the protein or the polypeptide of chemically modified, they by natural process as processing and other posttranslational modification and by chemically modified, also can be by chemical modification technology as adding one or more peg molecules, sugar, phosphoric acid salt and/or other this molecule, wherein this quasi-molecule is not the natural molecule that is incorporated into wild-type protein or S05A peptide.Derivative comprises salt.This chemically modified is described in visible basic material and more detailed monograph and the big quantity research document, and they are well-known to those skilled in the art.Should be understood that the modification that can have the identical or different same type of degree on some sites of given protein or polypeptide.Equally, given protein or polypeptide can comprise the modification of numerous species.Modification can occur in protein or polypeptide Anywhere, comprises peptide main chain, amino acid side chain and amino or C-terminal.Modification comprises for example acetylize; acidylate; the ADP-ribosylation; amidation; the covalent attachment of flavine; the covalent attachment of heme moiety; the covalent attachment of Nucleotide or nucleotide derivative; the covalent attachment of lipid or lipid derivate; the covalent attachment of phosphatidylinositols; crosslinked; cyclisation; disulfide linkage forms; demethylation; form covalent cross-linking; form halfcystine; form Pyrrolidonecarboxylic acid; formylation; γ-carboxylated; glycosylation; GPI anchor (anchor) forms; hydroxylation; iodate; methylate; myristoylation; oxidation; proteolysis processing; phosphorylation; isoprenylation; racemization; glycosylation; the lipid combination; sulfation; the γ of glutaminic acid residue-carboxylated; hydroxylation and ADP-ribosylation; selenoization (selenoylation); the amino acid of transfer RNA mediation adds protein, as arginylization and ubiquitinization.Referring to for example " protein-structure and molecular property " (Proteins-Structure And Molecular Properties), second edition, T.E.Creighton, (the T.E.Creighton of WHF company, W.H.Freeman and Company), New York (1993) and Wold, F., " post translational protein modification: viewpoint and prospect " (Posttranslational Protein Modifcation:Perspectiveand Prospects), publish in " proteinic translation back covalent modification " (Posttranslational ConvalentModification of Proteins), the 1-12 page or leaf, B.C.Johnson compiles, the academic press, New York (1983); Seifter etc., Meth.Enzymol.182:626-646 (1990) and Rattan etc., " protein synthesis: posttranslational modification and aging " (Posttranslational Modifcation and Aging), Ann.N.Y.Acad.Sci.663:48-62 (1992).Term " derivative " comprises that chemically modified causes protein or polypeptide to become branch-like or is with or without the ramose ring-type.Ring-type, branch-like and branch's cyclic protein matter or polypeptide may be produced and also can be produced by synthetic method fully by translation back natural process.

Term " homologue " refers to that being measured to aminoacid sequence according to the standard method that is usually used in the amino acid position similarity of comparison two peptide species has 60% identical protein at least with the aminoacid sequence of S05A peptide.Similarity or homogeny degree between two kinds of protein are not difficult to calculate with currently known methods, include but not limited to following described those methods: " calculating molecular biology " (Computational Molecular Biology), Lesk, A.M. compile, Oxford University Press (Oxford University Press), New York, 1988; " biological computation: information science and genome plan " (Biocomputing:Informatics and Genome Projiects), Smith, D.W. compiles, academic press, New York, 1993; " Computer Analysis of sequence data " (Computer Analysisof Sequence Data), part i, Griffin, A.M. and Griffin, H.G. compiles, Xiu Mana press (Humana Press), New Jersey, 1994); " molecular biology sequential analysis " (Sequence analysisin Molecular Biology), von Heinje, G., academic press, New York, 1987); " sequence analysis primer " (Sequence analysis Primery), Gribskov, M. and Devereux, J. compiles, MS press (M Stockton Press), New York, 1991; Carillo H. and Lipman, D., SIAM, J.AppliedMath., 48:1073 (1988).Design the preferred method of measuring homogeny and provided maximum match between the cycle tests.In the available computer program of the public, enrolled the method for measuring homogeny and similarity.

Be used for determining that the preferred computer program technic of homogeny and similarity includes but not limited to GCG routine package (Devereux between two kinds of sequences, J. etc., Nucleic Acids Research, 12 (1): 387 (1984)), BLASTP, BLASTN and FASTA, Atschul, S.F. etc., J.Molec.Biol., 215:403-410 (1990).The public can obtain BLASTX program (BLAST handbook, Altschul, S. etc., NCBI NLM NIH Bethesda, Md.20894 from NCBI and other source; Altschul, S. etc., J.Mol.Biol., 215:403-410 (1990).For example adopt computerized algorithm such as GAP (genetics computer set, state of Wisconsin university (University of Wisconsin), state of Wisconsin Madison (Madison, Wis.)), arrange the sequence homogeny per-cent that contrasts two kinds of protein to be determined or polypeptide and be used for their amino acid (algorithm is determined " matching range ") separately of optimum matching.

The open point penalty in room (gap opening penalty) (is calculated as the average diagonal lines of 3x; " average diagonal lines " is the diagonal averages of used comparator matrix; " diagonal lines " is scoring or the numerical value of giving each preferred amino acid by specific comparator matrix) and the room extend point penalty (gap extension penalty) (normally 1/10 of the open point penalty in room) and comparator matrix for example PAM250 or BLOSUM 62 therewith algorithm be used in combination.Algorithm also can use standard comparator matrix (the PAM250 comparator matrix is referring to Dayhoff etc., " protein sequence and structure atlas " (Atlas of Protein Sequence and Structure), the 5th volume, enlarged edition 3[1978]; BLOSUM 62 comparator matrixs are referring to Henikoff etc., Proc.Natl.Acad.Sci.USA, 89:10915-10919).Use this arithmetic calculation homogeny per-cent then.Homologue is compared with reference protein or peptide, has one or more aminoacid replacement, disappearance and/or insertion usually, depends on the circumstances.

Term " fusion rotein " refers to a kind of protein, wherein one or more peptide reorganization merge or chemical coupling (comprising covalently or non-covalently) in a protein, for example (but being not limited to) antibody or antibody fragment are as F _AbFragment or short chain Fv etc.Term " fusion rotein " also refers to the polymer (being the polymer of dimer, tripolymer, the tetramer and Geng Gao) of peptide.These polymers comprise the same poly-polymer that contains a kind of peptide, contain the different poly-polymer of more than one peptides and contain at least a peptide and at least a other proteinic different poly-polymers.These polymers can be the effects of hydrophobic, hydrophilic, ion and/or covalent effect, key or joint, can be to be cross-linked to form with linkers, maybe can be by for example, and form liposome and connect indirectly.

Term " peptide mimics " or " stand-in " refer to can simulating peptide or proteinic biological activity but no longer be the bioactive compounds of peptide on chemical property, and promptly they no longer contain any peptide bond (being the amido linkage between amino acid).Here the term peptide mimics is used for more broad sense, comprises it being molecule such as false peptide, half peptide and the plan peptide (peptoid) of peptide in nature no longer fully.The example of the peptide mimics of broad sense (wherein the part of peptide is lacked the structure replacement of peptide bond) is described below.No matter be fully or part is non-peptide, peptide mimics of the present invention provides the spatial disposition of reactive chemical part, and the three-dimensional arrangement of active group is extremely similar in the peptide of described part and peptide mimics institute foundation.Because the geometry of this similar avtive spot, peptide mimics is similar to the biological activity of this peptide to the effect of biosystem.

Peptide mimics of the present invention preferably is substantially similar to peptide as herein described on 3D shape and biological activity.Structurally modify peptide known in the art and comprise that with the example of the method that produces peptide mimics being inverted the main chain chiral centre produces D-amino-acid residue structure, specifically be at the N-end, thereby strengthened the stability of proteasome degradation and can influence activity sharply." contain tritium D-L-Ala at paper ¹-peptide T is in conjunction with (Tritriated D-ala.sup.1-Peptide T Binding) ", Smith C.S. etc., Drug DevelopmentRes., 15, an example has been described in the 371-379 page or leaf (1988).Second method is for stability changes ring texture, arrives C interchain imide and lactan (Ede etc., Smith and Rivier volume as N, " chemistry and biology of peptide " (Peptides:Chemistry and Biology), Escom, Leiden, 1991, the 268-270 page or leaf).Example is seen the thymopeptide-5 sample compound of configuration restriction, as U.S. Patent number 4,457, and 489 (1985), Goldstein, G. etc. are disclosed like that, and it is for reference that its content is all included this paper in.The third method is with the peptide bond in the false peptide bond replacement peptide that can give the proteolysis resistance.

Many false peptide bonds generally do not influence the structure and the biological activity of peptide.An example of this method is to replace converse (retro-inverso) false peptide bond (" the converse analogue of the biological activity of thymopeptide-5 ") (Biologically activeretroinverso analogue of thymopentin), Sisto A etc., Rivier, J.E. and Marshall, G.R. compile, " peptide; chemistry; structure and biology " (Peptides, Chemistry, Structure and Biology), Escom, Leiden, 1990,722-773 page or leaf) and Dalpozzo etc. (1993), Int.J.Peptide ProteinRes., 41:561-566, it is for reference to include this paper in).Modify according to this, the aminoacid sequence of peptide may be identical with the sequence of above-mentioned peptide, except one or more peptide bonds are replaced by converse false peptide bond.Preferred most of N-terminal peptide bond is substituted, because this replacement is to act on the N-end and can give the proteolysis resistance by exopeptidase.Also the chemical group of available other similar structures substitutes amino acid whose chemical group and does further modification.Another kind of known improve and biological activity does not have or the suitable false peptide bond of little loss is the false peptide bond of structure things such as reduced form (reduced isostere) (Couder etc. (1993) to enzyme cutting stability, Int.J.PeptideProtein Res., 41:181-184, it is for reference all to include this paper in).

Therefore, the aminoacid sequence of these peptides may be identical with the sequence of certain peptide, except one or more peptide bonds are substituted by false peptide bond such as thing such as row such as structure such as grade.Preferred most of N-terminal peptide bond is substituted, because this replacement is to act on the N-end and can give the proteolysis resistance by exopeptidase.Synthetic have structure thing such as one or more reduced forms arrange the peptide of false peptide bond be known in the art (Couder etc., (1993), above institute quote).Other example comprises that importing ketone methylene radical (ketomethylene) key or methyl sulfide (methylsulfide) key substitute peptide bond.

The plan peptide derivant of peptide has been represented another kind of peptide mimics, intends peptide and has kept bioactive important structure determinant, but remove peptide bond, thereby give proteolysis resistance (Simon etc., 1992, Proc.Natl.Acad.Sci.USA, 89:9367-9371, it is for reference all to include this paper in).Intending peptide is the oligomer of the glycine of N-replacement.Reported some N-alkyl groups, each corresponding to side chain of natural amino acid (Simon etc., 1992, above institute quote).The some or all of amino acid of peptide are used corresponding to alternative amino acid whose N-substituted glycinic acid and are substituted.

Term " peptide mimics " or " stand-in " also comprise anti--D peptide and the enantiomer that defines below.

Term " anti--D peptide " refers to compare with the L-aminoacid sequence of peptide, biological activity protein or peptide that the D-amino acid of being arranged by reverse sequence is formed.Therefore, the carboxyl terminal residue of L-amino acid peptide becomes the N-terminal of D-amino acid peptide, or the like.For example, the ETESH of this peptide becomes H _dS _dE _dT _dE _d, E wherein _d, H _d, Sd and Td be the D-amino acid that corresponds respectively to L-amino acid E, H, S and T.

Term " enantiomer " refers to a kind of biological activity protein or peptide, and wherein one or more L-amino-acid residues are substituted by corresponding D-amino-acid residue in the aminoacid sequence of peptide.

" composition " used herein broadly refers to contain the peptide enumerated or any composition of aminoacid sequence.Said composition can comprise dry preparation, the aqueous solution or sterilization composition.Can utilize the composition that contains peptide as hybridization probe.This probe can be preserved by lyophilized form, and can add stablizer such as carbohydrate.During hybridization, this probe can for example use in the aqueous solution of Denhardt solution, dried milk, salmon sperm dna etc. in saliferous such as NaCl, washing composition such as sodium lauryl sulphate (SDS) and other component.

The present invention relates to contain composition just like the above defined S05A peptide of the present invention.

In addition, the present invention includes other protein that contain all or part of S05A peptide, thus this protein preferably has identical with this peptide, similar or enhanced biological activity.Adopt the guidance that this paper provided, the those skilled in the art in this field can be according to the synthetic specified protein of the aminoacid sequence of any S05A peptide, and described S05A peptide finds it is to cause the potent agent of necrocytosis and tested them as the effectiveness that causes necrocytosis reagent.

Other peptide sequence derived from S05A peptide (finding that they are the potent agents that cause necrocytosis) also is the potent agent that causes necrocytosis.According to the instruction of this paper, the those skilled in the art in this field need not undo experimentation, just can synthesize the fragment of effective peptide of crossing over this proteinic whole aminoacid sequence, so that identify other effective peptide sequence.

S05A peptide in the particularly preferred embodiment comprise include, but not limited to following these:

SEQ ID NO.2 IDQQVLSR IIle-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile

SEQ ID NO.3 KLEIKRCL Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu

SEQ ID NO.4 VLSRIK Val-Leu-Ser-Arg-Ile-Lys

SEQ ID NO.5 RIKLEIK Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg

SEQ ID NO.6 VLSRIKLEIKRCL Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu

SEQ ID NO.7 IDQQVLSRIKLEI Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile

Those skilled in the art can understand, other that can select above-mentioned S05A peptide be than small segment, and make these fragments have same or analogous biologic activity.Those skilled in the art can select other fragments, and make these peptides have same or analogous biologic activity.Peptide of the present invention comprises these other fragments.Peptide generally speaking of the present invention has at least 6 amino acid, preferably at least 5 amino acid, more preferably at least 4 amino acid.

The present invention also comprises the S05A peptide that contains two or many S05A peptides that link together.As long as a S05A peptide has required biological activity, two such peptides also have required biological activity.

S05A peptide and fragment, variant, derivative, homologue, fusion rotein and stand-in that available method known to those skilled in the art preparation the present invention includes, for example peptide, albumen, AD7c-albumen and its fragment, variant, derivative and the homologue of recombinant DNA technology, protein synthesis and separating natural generation.

Adopt method known to those skilled in the art, can prepare S05A peptide and fragment, variant, derivative, homologue, fusion rotein and stand-in from other peptide, protein and fragment thereof, variant, derivative, homologue.These methods include, but is not limited to proteolytic enzyme peptide or albumen be cut into required S05A peptide.

The S05A peptide can prepare with the recombinant DNA technology method of knowing, as list in Sambrook etc., " molecular cloning: laboratory manual " (Molecular Cloning:A Laboratory Manual), press of cold spring harbor laboratory (Cold Spring Harbor Laboratory Press), cold spring port, New York (Cold SpringHarbor, and/or " newly organized molecular biology experiment guide " (CurrentProtocols in Molecular Biology) of compiling such as Ausubel N.Y.)), company limited of Green press (Green Publishers Inc.) and Willie father and son company (Wiley and Sons), the method for New York (N.Y.).

Can pass through, for example screening-gene group or cDNA library or obtain the gene or the cDNA of coding S05A peptide by pcr amplification.Being used to screen the probe in library or primer can produce as the conservative motif of finding in other peptide or albumen according to the sequence information of the gene fragment of other known or identical or genes involved family.In addition, if identified the gene of coding S05A peptide, all or part of probe that can be used as of this gene is to identify homologous gene.Probe or primer can be used for screening the cDNA library from thinking to express S05A peptide gene multiple tissue-derived.Usually, can use high preciseness condition to reduce the number of false positives that screening obtains as far as possible.

The method of the gene of another kind of preparation coding S05A peptide adopts chemosynthesis, and the method that use technology personnel know is as (Angew.Chem.Intl.Ed., 28:716-734) methods of Miao Shuing such as Engels.These methods comprise that methods such as phosphotriester, phosphoramidite, H-phosphonic acid ester come nucleic acid.The preferred method of this chemosynthesis is to use polymkeric substance-support synthetic of standard phosphoramidite chemistry.Generally, the DNA length of encoded peptide is a hundreds of Nucleotide.Available these methods will synthesize several fragments greater than the nucleic acid of about 100 Nucleotide.Fragment can be joined together to form the peptide or the protein of total length then.Usually, the aminoterminal dna fragmentation of coded protein has the ATG of coding methionine(Met).This methionine(Met) can or can not be present in the mature form of albumen or peptide, and this depends on whether the protein that produces in the host cell is designed to secrete from this cell.

Gene, cDNA or its fragment available standards interconnection technique of coding S05A peptide are inserted in suitable expression or the amplification vector.Usually be chosen in carrier that function is arranged in the used concrete host cell (be that carrier is compatible with host cell mechanism, thereby amplifiable gene and/or expressing gene).Gene, cDNA or its fragment of coding S05A peptide can amplification/expression in protokaryon, yeast, insect (rhabdovirus system) and/or eukaryotic host cell.The selection of host cell depends in part on S05A peptide whether glycosylation and/or phosphorylation.If preferred yeast, insect or mammalian host cell like this.

Usually, used carrier comprises at least one 5 ' flanking sequence (being also referred to as " promotor ") in any host cell, also comprise other regulatory element, for example the starting point of enhanser, reproduction element, Transcription Termination element, the complete intron sequences that contains donor and acceptor splice site, signal peptide sequence, ribosome bind site element, polyadenylation sequence, be used to insert polylinker zone, selectable identification element that coding is treated the nucleic acid of express polypeptide.These elements hereinafter have been discussed separately.Optional is that carrier can comprise sequence label, promptly is positioned at the oligonucleotide molecules of 5 ' or 3 ' end of albumen or peptide-coding sequence; Oligonucleotide molecules coding polyhistidine (PolyHis) (as six Histidines (HexaHis)) or other label such as FLAG, HA (influenza virus hemagglutinin) or have the myc of commercialization antibody.This label is fused to polypeptide when being everlasting express polypeptide, thereby can be used as the method for the albumen or the peptide of affinity purification host cell.The antibody that for example can use anti-this label is finished affinity purification as affinity matrix by column chromatography.Randomly, subsequently by several different methods as from the albumen of purifying or peptide, removing label with some peptase.

Those skilled in the art can be merged human normal immunoglobulin hinge and Fc district at the N-or the C-end of S05A peptide.Available subsequently A albumen affinity column purifying Fc fusion rotein.The pharmacokinetics long half time of performance and finding merges transformation period that the protein of Fc shows in vivo significantly greater than fused protein not in the known Fc body.Equally, merge and make the molecule dimerization/multimerization useful to the Fc district the biological activity of some molecules.

5 ' flanking sequence can be homology (promptly from kind identical with host cell and/or bacterial strain), allos (promptly from kind different with host cell or bacterial strain), heterozygosis (promptly from 5 ' the flanking sequence combination that surpasses a source), synthetic, maybe can be native protein or peptide gene 5 ' flanking sequence.Equally, the source of 5 ' flanking sequence can be unicellular protokaryon or eukaryote, any vertebrates or invertebrates or any plant, as long as 5 ' flanking sequence has function and can be activated by host cell mechanism.

5 ' useful in the carrier of the present invention flanking sequence can obtain by the several method of knowing in any this area.Usually, the useful 5 ' flanking sequence of this paper except albumen or peptide gene flanking sequence is former obtains identifying that by mapping and/or digestion with restriction enzyme therefore available suitable restriction enzyme separates from suitable tissue-derived.In some cases, the full nucleotide sequence of 5 ' flanking sequence can be known.Here the synthetic 5 ' flanking sequence of available above-mentioned nucleic acid the method synthetic or clone.

As the whole of 5 ' flanking sequence or when only part is known, 5 ' flanking sequence fragment of the oligonucleotide that its available usefulness is suitable and/or identical or another kind adopts PCR and/or obtains by screening-gene group library.

If do not know 5 ' flanking sequence, can from comprise encoding sequence for example or even another kind of gene or several genes than separating the dna fragmentation that contains 5 ' flanking sequence the large fragment DNA.Available one or more careful enzymes of selecting are finished separation by digestion with restriction enzyme, thereby are separated suitable dna fragmentation.After the digestion, required fragment can pass through the sepharose purifying, Post or other method known to the skilled are separated.Selecting suitable enzyme is obvious to reach this purpose to the those skilled in the art in this field.

The starting point of reproduction element is the part of the commercial prokaryotic expression carrier of buying normally, and it assists the carrier amplification in the host cell.In some cases, carrier certain copy number that increases is important to optimum expression albumen or peptide.If selected carrier does not comprise the starting point of replication site, can and connect into carrier according to known array chemosynthesis starting point.The Transcription Termination element generally is positioned at 3 ' end of albumen or peptide-coding sequence, and it is used to stop transcribing of albumen or peptide.Usually, the Transcription Termination element in the prokaryotic cell prokaryocyte is that the back has the G-C of poly-T sequence to enrich fragment.Though can also be not difficult to synthesize with above-mentioned nucleic acid synthetic method from library clone or as the commercial element of buying of carrier part.

Selectable marker gene component numbering host cell is survived in selective medium and is grown necessary protein.The albumen mass-energy (a) of typical selectable marker gene component numbering is given the resistance of prokaryotic host cell to microbiotic or other toxin, and as ampicillin, tsiklomitsin or kantlex, (b) auxotroph of additional cell lacks; Or the crucial nutrition that (c) provides complex medium not have.But preferred selective marker is kalamycin resistance gene, ampicillin resistant gene and tetracycline resistance gene.

The rrna binding member that is commonly referred to as SD sequence (Shine-Dalgamo sequence) (prokaryotic organism) or Kozak sequence (eucaryon nuclear is biological) is normally essential to the mRNA translation initiation.This element is connected in 5 ' of 3 ' end of promotor and albumen to be synthesized or peptide-coding sequence usually and holds.The SD sequence is different but generally be poly-purine (being A-G content height).Identified many SD sequences, be not difficult to synthesize each sequence and can be used for prokaryotic vector with aforesaid method.

Needing the S05A peptide from the situation of secretory host cell, can use signal sequence to instruct peptide to arrive outside the host cell that synthesizes it, and can delete proteinic C-terminal part to prevent the film grappling.Usually, signal sequence is positioned at the coding region of S05A peptide gene or cDNA, or direct 5 ' end in S05A peptide gene coding region.Identified many signal sequences, any have the sequence of function to be used in combination with the gene or the cDNA of this peptide in selected host cell.Therefore signal sequence can with gene or the cDNA homology or the allos of this peptide, and can with gene or the cDNA homology or the allos of this peptide.In addition, signal sequence can be used the aforesaid method chemosynthesis.In most of situation, the existence by signal peptide is removed from the secretory host cell polypeptide can cause the N-terminal methionine(Met) of polypeptide.

In many cases, transcribing of S05A peptide gene or cDNA can increase by there being one or more introns in the carrier; When the S05A peptide at eukaryotic host cell, especially true when particularly producing in the mammalian host cell.Used intron can be natural generation in the S05A peptide gene, particularly when used gene is full-length gene group sequence or its fragment.When intron not in gene during natural generation (for most of cDNAs), intron can be originated available from another.Because intron must be transcribed effectively, the position of relative flanking sequence of intron and S05A peptide gene generally is important.Equally, the S05A peptide gene in inserting expression vector is that cDNA divides the period of the day from 11 p.m. to 1 a.m, and the optimum position of intron is transcription initiation site 3 ' end and many A transcription termination sequence 5 ' end.For the cDNA of this peptide preferably, one or more introns are positioned at cDNA one side or opposite side (promptly 5 ' or 3 ') thereby make it not interrupt this encoding sequence.If the host cell of intron and its insertion is compatible, any intron in any source can be used for putting into practice the present invention, and the source comprises any virus, protokaryon and eucaryon (plant or animal) biology.This paper also comprises synthetic intron.Randomly, in carrier, can use and surpass one intron.

When above-mentioned one or more elements were not present in the carrier to be used, they can obtain and connect into carrier separately.The method that is used to obtain each element is that the technician knows, and suitable with aforesaid method (being synthetic DNA, library screening etc.).

Being used to put into practice final carrier of the present invention can be from initial vector, as the vector construction of commerce purchase.This carrier can comprise or not comprise some elements that are included in the complete vector.If there is not required element to be present in the initial vector, can be compatible with the carrier end for being connected by make element end to be accessed with suitable restriction enzyme cut vector, thereby each element is connected into carrier separately.In some cases, must make the end that will connect become " flush end " to obtain satisfied connection.Obtain flush end by at first when all four kinds of Nucleotide exist, filling up " cohesive end " with KlenowDNA polysaccharase or T4DNA polysaccharase.This process is described in (the same) such as for example Sambrook to some extent well known.In addition, the two or more elements that are inserted into carrier can at first connect (if their position is adjacent one another are), connect into carrier subsequently.

The method of another kind of carrier construction is carried out all connections of multiple element simultaneously in a kind of reaction mixture.Here, because the inappropriate connection of element or insertion produce many nonsenses and no function carrier, yet can identify and the selection function carrier by digestion with restriction enzyme.

Putting into practice preferred vector of the present invention is those carriers compatible with bacterium, insect and mammalian host cell.This carrier comprises pCRII, pCR3 and pcDNA3.1 (San Diego, California hero company) InvitrogenCompany, San Diego, Calif.)), (the Zhuo Lasi Tu Te (StratageneCompany of genome company is drawn in the California to pBSII, La Jolla, Calif)), the pET15b ((Novagen of state of Wisconsin Madison Nuo Fugen company, Madison, Wis.)), the PGEX ((PharmaciaBiotech of New Jersey Piscataway Pharmacia biotech company, Piscataway, N.J.)), pEGFP-N2 (handkerchief Lip river, the California atropic clone technology (Clontech of company limited, Palo Alto, Calif.)), pETL (BlueBacII; Hero company) and pFastBacDual (Gibco/BRL, the special island (Grand Island, N.Y.)) of New York Glan.

After the nucleic acid molecule of the albumen of vector construction and will encode total length or brachymemma or peptide inserted the suitable site of carrier, complete vector can insert proper host cell and be used for amplification and/or expression of polypeptides.Host cell can be prokaryotic host cell (as intestinal bacteria) or eukaryotic host cell (as yeast cell, insect cell or vertebrate cells).When host cell is cultivated under conditions suitable, but synthetic proteins or peptide, (if host cell is secreted into it in substratum) or the directly collection from the host cell (if it is not secreted) that generates it from substratum subsequently of this albumen or peptide.

After the collection, method purifying such as S05A peptide useful molecules sieve chromatography, affinity chromatography.The host cell that select to produce albumen or peptide depend in part on this peptide whether glycosylation or phosphorylation (preferred eukaryotic host cell in this situation), host cell protein folding can be become its natural tertiary structure (as the suitable direction of disulphide bridges etc.) thus mode prepare biological activity protein by the bioactive peptide of tool, folding this peptide of the available appropriate chemical condition of discussing below in synthetic back.Suitable cell or clone can be mammalian cells, as Chinese hamster ovary cell (CHO), human embryo kidney (HEK) 293 or 293T cell or 3T3 cell.The method of mammalian host cell that selection known in the art is suitable and conversion, cultivation, amplification, screening and product generation and purifying.Other suitable mammal cell line is monkey COS-1 and COS-7 clone and CV-1 clone.Other exemplary mammalian host cell comprises primate cell system and rodent cells system, comprises cell transformed system.Normal diploid cell, derived from cultured cells strain outside the elementary organizer and former generation explant also be suitable.Candidate cell can be to select the genotype defective in the gene maybe can comprise the open gene of selecting of dominance.Other suitable mammal cell line includes but not limited to mouse neuroblastoma N2A cell, HeLa cell, mouse L-929 cell, the 3T3 system derived from Swiss, Balb-c or NIH mouse, BHK or HaK hamster cell system.

Be suitable for host cell of the present invention bacterial cell equally usefully.For example, multiple coli strain (as HB101, DH5 α, DH10 and MC1061) is the host cell of knowing in the biological technical field.The multiple bacterial strain of subtilis (B.subtilis), Rhodopseudomonas (Pseudomonas spp.), other bacillus (Bacillus spp.), streptomyces (Streptomyces spp.) etc. can be used for this method.Many yeast cell bacterial strains well known by persons skilled in the art also can be used as the host cell of expressing polypeptide of the present invention.

Insect cell system can be used for method of the present invention when needing in addition.For example Kitts etc. (Biotechniques, 14:810-817), Lucklow (Curr.Opin.Biotechnol., 4:564-572) and Lucklow etc. (J.Virol. has described this system in 67:4566-4579).Preferred insect cell is a Sf-9 and Hi5 (California Ka Ersibaide hero company (Invitrogen, Carlsbad, Calif.)).

Inserting (be also referred to as and transform or transfection) carrier methods such as available for example calcium chloride, electroporation, microinjection, fat transfection or DEAE-dextran method in the selected host cell realizes.The method of selecting partly is the function of host cell type to be used.These methods and other appropriate method are that the technician knows, and for example are seen in Sambrook etc., and be the same.

Containing the standard medium that the host cell available techniques personnel of carrier (promptly transform or transfection) know cultivates.Substratum comprises all cells growth and the essential nutrition of surviving usually.The suitable culture medium of cultivating Bacillus coli cells is for example Luria Broth (LB) and/or Terrific Broth (TB).Cultivating eukaryotic suitable culture medium is RPMI1640, MEM, DMEM, and they all can add serum and/or cultivate specific cells is required somatomedin.The suitable culture medium of cultivating insect (cell) is the Grace substratum, and it optionally is added with essential yeast extract (yeastolate), opalescin hydrolysate and/or foetal calf serum.Usually, microbiotic or other compound that only is used for the selective growth transformant add substratum as additive.The identification element the selected decision that exists on the plasmid of compound to be used by transformed host cell.For example, in the time can selecting identification element to be kalamycin resistance, the compound that adds substratum is a kantlex.

The available standard method assessment known in the art of the amount of the S05A peptide that produces in the host cell.This method includes but not limited to western blot analysis, SDS-polyacrylamide gel electrophoresis, native gel electrophoresis, HPLC separation, mass spectrum, immunoprecipitation and/or activity test such as DNA attached gel displacement test.

If albumen or peptide are designed to secrete from host cell, most of albumen or peptide can be found in cell culture medium.Protein with this method preparation does not have the N-terminal methionine(Met) usually, because it is removed when secreting from cell.If yet albumen or peptide from host cell, do not secrete, it can be present in tenuigenin and/or the nuclear (eukaryotic host cell) or in cytosol (Gram-negative bacteria host cell), and can have the N-terminal methionine(Met).

Be located in the S05A peptide in host cell tenuigenin and/or the nuclear, usually at first mechanically or with the broken host cell of washing agent with composition in the release born of the same parents in buffered soln.From then on separate this peptide in the solution then.

Purifying S05A peptide can be finished with multiple technologies from solution.If synthetic protein contains label as six Histidines (for example peptide/six Histidines) or other little peptide such as FLAG (St. Louis, the state of Michigan (Sigma-Aldritch of Sigma company, St.Louis, MI)) or at its carboxyl or N-terminal contain calmodulin-binding peptide (Zhuo Lasi Tu Te genome company is drawn in the California), basically can make solution pass affinity column by one step process and come purifying, wherein base for post confrontation label or directly protein is had high-affinity (being the monoclonal antibody of this peptide of specific recognition).For example, polyhistidine with high-affinity and specificity in conjunction with nickel, zinc and cobalt; Thereby use the fixing metal ions affinity chromatography of the affine resin of nickel (as being used for the QIAexpress system of proper root company (Qiagene) or the Xpress system of hero company) or the affine resin of cobalt (as be used for BD Biosciences-CLONTECH Talon system) to can be used for purified peptide/polyhistidine.(referring to for example, " newly organized molecular biology experiment guide " 10.11.8 chapter that Ausubel etc. compile, (the John Wiley ﹠amp of John Willie father and son company; Sons), New York).

When the S05A peptide of preparation combination tag and when not having antibody and can use not, the purification process that can use other to know.These methods include but not limited to the natural gel electrophoresis and the preparation type isoelectrofocusing (Isoprime machine/technology, Hou Fu scientific ﹠ technical corporation (Hoefer Scientific)) of ion exchange chromatography, hydroxyapatite, hydrophobic interaction chromatography, sieve chromatography, HPLC, attached gel wash-out.Under the certain situation, can be with two or more combinations in these technology to obtain higher degree.

If expection S05A peptide mainly is found in the born of the same parents, the known any standard technique of available techniques personnel is extracted intracellular organic matter (inclusion body that comprises Gram-negative bacteria) from host cell.For example can pass through French press, homogenization and/or supersound process, centrifugally then come the cracking host cell to discharge pericentral siphon/cytoplasmic inclusion.If this peptide forms inclusion body in cytosol, inclusion body usually can be in conjunction with interior and/or outer cytolemma, mainly finds in sedimentable matter after therefore centrifugal.Can or reductive agent arranged at extreme pH then, handle sedimentable matter to discharge, to rupture and the dissolving inclusion body with chaotropic agent such as washing agent, guanidine, guanidine derivative, urea or urea derivative down as the dithiothreitol (DTT) of alkaline pH or the three propyloic phosphines existence of acid pH.Analyses such as available then gel electrophoresis, immunoprecipitation are in the peptide of soluble form at present.Separate this peptide if desired, the available standards method is finished separation, as hereinafter with (Meth.Enz., the methods of 182:264-275) listing such as Marston.

In some cases, the S05A peptide can not have biological activity after separating.Can adopt again several different methods folding or that polypeptide changed into its tertiary structure and produce disulfide linkage to recover biological activity.These methods comprise is exposed to usually above among 7 the pH and have a chaotropic agent of specific concentrations the dissolved polypeptide.The selection of chaotropic agent is very similar to the selection that is used to dissolve inclusion body, but usually concentration is lower and may not be the used identical chaotropic agent of dissolving.In most of situation, the reductive agent that folding again/oxidizing solution also comprises reductive agent or specified proportion adds its oxidised form, makes disulphide reorganization form proteinic halfcystine bridge to produce into specific redox-potential.Some redox couples commonly used comprise halfcystine/cystamine, the two GSH (dithiobis GSH) of gsh (GSH)/two sulphur, cupric chloride, dithiothreitol (DTT) (DTT)/dithiane DTT, 2 mercapto ethanol (bME)/two sulphur-b (ME).In many cases, need cosolvent to improve folding again efficient, be used for the how conventional reagent of this purpose and comprise glycerine, different molecular weight polyethylene glycol and arginine.

If do not form the S05A peptide inclusion body of significance degree in the host cell, the centrifugal back of cell homogenates finds that the S05A peptide mainly is arranged in supernatant liquor, and the S05A peptide can separate from supernatant liquor with following method.

Preferably partially or completely separating in the situation of S05A peptide, purifying is finished in the standard method that available techniques personnel know.These methods include but not limited to electrophoretic separation, then electroelution, various types of chromatography (immunity is affine, molecular sieve and/or ion-exchange) and/or high pressure liquid chromatography (HPLC).In some cases, the preferred use surpasses the next complete purifying of these a kind of methods.

Except preparing and purifying S05A peptide with recombinant DNA technology, S05A peptide and their fragment, variant, homologue, fusion rotein, peptide mimics and derivative can use technology known in the art by chemical synthesis process preparation (synthetic as solid-phase peptide), (J.Am.Chem.Soc. such as Merrifield for example, 85:2149), (Proc.Natl.Acad.Sci.USA such as Houghten, 85:5132), Stewart and Young (" solid-phase peptide is synthetic " (Solid Phase Peptide Synthesis), Illinois Rockford Pierre Si chemical company (Pierce Chemical Co., Rockford, IL)) described in.Can synthesize this peptide that is with or without methionine(Met) at N-terminal.The listed method oxidation of S05A peptide available reference document of chemosynthesis is to form disulphide bridges.Expection S05A peptide have can produce with reorganization or purifying from the suitable biological activity of the peptide of natural origin, therefore can use with reorganization or native peptides exchange.

Wherein the chemically modified S05A peptide combinations that links to each other with polymkeric substance of peptide is included in the scope of the present invention.Thereby the common water soluble of selected polymkeric substance makes and its bonded protein can be at aqueous environments, as precipitating in the physiological environment.Selected polymkeric substance is modified usually having single reactive group as active ester that is used for acidylate or the aldehyde that is used for alkanisation, thereby can be as the controlled polymerization degree that present method provided.Polymkeric substance can be any molecular weight, can be branch or branch not.Be included in the peptide polymer scope is mixture of polymers.

In some cases, the nucleic acid and/or the amino acid variant that need the natural generation S05A peptide of preparation.The nucleic acid variant can use site-directed mutagenesis, pcr amplification or other appropriate method to produce, and wherein primer has required point mutation (description of induced-mutation technique is referring to Sambrook etc., and is the same, and Ausubel etc. are the same).(the same) described chemical synthesis process such as use Engels also can be used for preparing this variant.Other method known to the skilled also can be used.

Preferred nucleic acid variant is included in those nucleic acid of the Nucleotide replacement that causes codon preference in the host cell that produces the S05A peptide.This " codon optimization " can be determined by computerized algorithm, the sub-frequency meter of described algorithm combining cipher as " Ecohigh.Cod " as the codon preference of highly expressing bacterial gene, the state university in Wisconsin, state of Wisconsin Madison city 9.0 version software bags for example, the genetics computer set provides.Other useful codon frequency table comprises " Celegans_high.cod ", " Celegans_low.cod ", " Drosophila_high.cod ", " Human_high.cod ", " Maize_high.cod " and " Yeast_high.cod ".Other preferred variants is that coding is compared the variant of above-mentioned conservative amino acid variation (as wherein the electric charge or the polarity of natural generation amino acid side chain significantly do not change because of the replacement with different aminoacids) and/or the variant that is designed to produce the variant of new glycosylation and/or phosphorylation site or is designed to delete existing glycosylation and/or phosphorylation site with wild-type.

The known conventional peptide synthetic technology of S05A peptide and fragment thereof, homologue, variant, fusion rotein, peptide mimics, derivative and salt available techniques personnel produces.These technology comprise that the chemical coupling method is (referring to Wunch, E: " organic chemistry method " (Methoden der organischen Chemie), the 15th volume, the 1+2 district, " peptide is synthetic " (Synthese von Peptiden), thime Verlag, Stuttgart (Stuttgart) (1974) and Barrany, G.; Marrifield, R.B.: " peptide " (The Peptides), E.Gross, J.Meienhofer compiles, the 2nd volume, the 1st chapter, the 1-284 page or leaf, academic press (1980)), the enzyme coupling method (referring to Widmer, F.Johansen, J.T., Carlsverg Res.Commun., the 44th volume, 37-46 page or leaf (1979) and Kullmann, W.: " the enzyme peptide is synthetic " (Enzymatic Peptide Synthesis), company of CRC press (CRC PressInc.), Boca Raton, Fla. (1987) and Widmer, F., Johansen, J.T., " the synthetic peptide in biology and the medical science " (Synthetic Peptides in Biology and Medicines), Alitalo, K., Partanen, P., Vatieri, A. compile 79-86 page or leaf, Elsevier, Amsterdam (1985)), or the combination of chemistry and enzyme method, if this has superiority for method design and economy.Use guide provided herein and instruction, the peptide sequence that those skilled in the art can change the S05A peptide has identical with original or natural S05A peptide or similar bioactive homologue with generation.

Use stand-in rather than this peptide itself of given S05A peptide to have advantage.Usually, compare with peptide with protein, the bioavailability of peptide mimics is higher, and action time is longer and production cost is lower.

Therefore, thus above-mentioned peptide can be used for developing and has the little chemical compound that similar biological activity has similar therapeutic action.The peptide mimics that can use combinatorial chemistry technique and other technological development S05A peptide known in the art is (referring to for example " the 20th European peptide symposium record " (Proceedings of the 20thEuropean Peptide Symposium), G.Jung, E.Bayer compiles, 289-336 page or leaf and reference wherein).

Modified peptides comprises that with the example of the means known in the art that produce peptide mimics being inverted the main chain chiral centre produces D-amino-acid residue structure on the structure, specifically is at the N-end, thereby has strengthened proteasome degradation stability and influence is active unfriendly." contain tritium D-L-Ala 1-peptide T combination " (TritriatedD-ala.sup.1-Peptide T Binding) at paper, Smith C.S. etc., Drug Development Res., 15, provided an example in the 371-379 page or leaf (1988).

Second method is to change the required ring texture of stability, arrives C interchain imide and lactan (Ede etc., Smith and Rivier volume as N, " peptide: chemistry and biology " (Peptides:Chemistryand Biology), Escom, Leiden, 1991, the 268-270 page or leaf).Example is seen the thymopeptide-5 sample compound of configuration restriction, as U.S. Patent number 4,457, and 489 (1985), Goldstein, G. etc. are disclosed like that, and it is for reference that its content is all included this paper in.

The third method is with the peptide bond in the false peptide bond replacement S05A peptide that can give the proteolysis resistance.General structure and the bioactive many false peptide bonds that does not influence peptide has been seen in description.An example of this method is to replace converse false peptide bond (" the converse homologue of the biological activity of thymopeptide-5 " (Biologically activeretroinverso analogues of thymopentin), SistoA etc., Rivier, J.E. and Marshall, G.R. compile, " peptide, chemistry, structure and biology " (Peptides, Chemistry, Structure and Biology), Escom, Leiden, 1990,722-773 page or leaf) and Dalpozzo etc. (1993), Int.J.Peptide ProteinRes., 41:561-566, it is for reference to include this paper in).Modify according to this, the aminoacid sequence of peptide may be identical with the sequence of above-mentioned peptide, except one or more peptide bonds are replaced by converse false peptide bond.Preferred most of N-terminal peptide bond is substituted, because this replacement is to act on the N-end and can give the proteolysis resistance by exopeptidase.

The peptide of the synthetic converse false peptide bond of the one or more reduced forms of tool is in ((1993) such as Sisto (1990) and Dalpozzo, above quote) known in the art.Therefore peptide bond can substitute with non-peptide bond and make peptide mimics obtain the structure similar to original peptide, thereby biological activity is similar.Also the chemical group of available other similar structures substitutes amino acid whose chemical group and does further modification.Another kind of known improve and biological activity does not have or the suitable false peptide bond of little loss is the false peptide bond (Couder etc. (1993) of structure thing such as reduced form to the stability of enzyme cutting, Int.J.Peptide Protein Res., 41:181-184, it is for reference all to include this paper in).Therefore, the aminoacid sequence of these peptides may be identical with the sequence of certain peptide, except one or more peptide bonds are substituted by false peptide bond such as thing such as structure such as grade.Preferred most of N-terminal peptide bond is substituted, because this replacement is to act on the N-end and can give the proteolysis resistance by exopeptidase.Synthetic peptide with false peptide bond of structure thing such as one or more reduced forms is (Couder etc., above institute quote) known in the art.Another example comprises that importing ketone methene key or methyl sulfide key substitute peptide bond.

The plan peptide derivant of S05A peptide has been represented another kind of peptide mimics, intend peptide and kept bioactive important structure determinant, but removal peptide bond, thereby give proteoclastic resistance (Simon etc., 1992, Proc.Natl.Acad.Sci.USA, 89:9367-9371, it is for reference all to include this paper in).Intending peptide is the oligomer of the glycine of N-replacement.Reported some N-alkyl groups, each corresponding to side chain of natural amino acid (Simon etc., 1992, above institute quote).The some or all of amino acid of this peptide are used corresponding to alternative amino acid whose N-substituted glycinic acid and are substituted.

Can determine that the tertiary structure of original peptide assists to develop peptide mimics by NMR spectroscopy, crystallography and/or computer-auxiliary molecule modeling.These technology help exploitation to have to compare with original peptide effectiveness higher and/or bioavailability is higher and/or stable higher novel composition (Dean (1994), BioEssays, 16:683-687; Cohen and Shatzmiller (1993), J.Mol.Graph., 11:166-173; Wiley and Rich (1993), Med.Res.Rev., 13:327-384; Moore (1994), Trends Pharmacol.Sci., 15:124-129; Hruby (1993), Biopolymers., 33:1073-1082; Bugg etc. (1993) Sci.Am., 269:92-98, it is for reference all to include this paper in).

In case a kind of potential peptide mimics compound identified, it is can be with generalized method in the following example synthetic and analyze to assess its activity.Belong to the scope of the invention with the biological activity with peptide of aforesaid method acquisition and the peptide simulated compound of similar three-dimensional structure.It will be apparent to one skilled in the art that peptide mimics can produce from any peptide that carries one or more above-mentioned modifications.More clearly, peptide mimics of the present invention can be further used for developing more effective non-peptide compound except the effectiveness as the treatment compound.

Today, many mechanisms can both synthesize peptide as herein described.For example, obtain a kind of sequence of S05A peptide, these mechanisms just can synthesize peptide and provide the synthetic peptide and the enclose file and the evidence of peptide identity.

The present invention comprises that also use S05A peptide and their corresponding nucleic acids molecules are used for test, whether to have S05A peptide, S05A peptide DNA or corresponding RNA in qualitative or quantitative test mammalian tissues or the humoral sample.S05A peptide and their corresponding nucleic acids molecules can be used for the preparation of these tests, and no matter whether the coding DNA of this peptide or this peptide shows biological activity.The S05A peptide nucleic acid sequence can be used as useful hybridization probe source whether to have the DNA or the corresponding RNA of this peptide in qualitative or quantitative test mammalian tissues or the humoral sample.Itself not the bioactive S05A peptide nucleic acid sequence of tool can be used to prepare identification and/or in conjunction with the antibody of S05A peptide.This antibody available standards method preparation.Therefore, short chain antibody fragment and other fragment reaction with S05A reactive polypeptide or bonded antibody and this antibody also belongs to the scope of the invention.Antibody can be polyclone, mono-clonal, reorganization, chimeric, strand and/or dual specific.Usually, antibody or its fragment are that the people originates or " humanization ", promptly are prepared into giving the immune response that patient Shi Neng prevented or reduced as far as possible antagonist.Preferred antibody is people's antibody, no matter polyclone or mono-clonal.Antibody fragment can be any fragment with reactive polypeptide of the present invention, as Fab, Fab ' etc.The present invention also provides hybridoma, and it is by giving selected Mammals with any S05A peptide as antigen presentation, then merges mammiferous cell (as splenocyte) and some cancer cells to produce immortalized cell and be and to produce with known technology.The method that produces the antibody of this clone and anti-all or part S05A also belongs to the scope of the invention.

Antibody also can be used in the body and in-vitro diagnosis or research purpose, as detect whether there is the S05A peptide in body fluid or the cell sample with mark pattern.

The present invention also comprises one or more S05A peptides in test as the application of verification standard substance, whether has S05A peptide, albumen, peptide DNA, protein dna or corresponding RNA in described test qualitative or quantitative test mammalian tissues or the humoral sample.

The present invention relates to treat the method for the illness that need remove cell, as optimum or malignant tumour, gland (as prostate gland) hyperplasia, unwanted facial hair, wart and unwanted fatty tissue, or suppress or prevent unwanted cells propagation, as the restenosis of support (stent).This method comprise will the treatment significant quantity the S05A peptide Mammals of these needs is arranged, or for example be used to be coated with device such as support.

Described illness can be, for example, the tumour of lung, mammary gland, stomach, pancreas, prostate gland, bladder, bone, ovary, skin, kidney, hole, colon, intestines, stomach, rectum, esophagus, blood, brain and its coverture, spinal cord and its coverture, muscle, reticular tissue, suprarenal gland, Parathyroid, Tiroidina, uterus, testis, hypophysis, reproductive organ, liver, gall-bladder, Eye Ear Nose And Throat, tonsil, mouth, lymphoglandula and lymphsystem and other organ.

As used herein, term " malignant tumour " comprises human carcinomas, sarcoma and melanomatous form of ownership, and they occur with minuent differentiation, appropriateness differentiation, well differentiated form.

The present invention satisfies this area, and risk is lower for removing innocent tumour and the less treatment needs of adverse side effect of operation.Need to remove the carcinoid method in deep (for example brain, heart, lung etc.) in operation hazardous area such as the body especially.

Treatment must remove cell illness method can with the treatment these illnesss ordinary method, be used in combination as excision, chemotherapy and radiation.Peptide can before these conventional treatmenies, during or use afterwards.

Illness to be treated also can be hamartoplasia, hypertrophy or hypertrophy, and described tissue is selected from: lung, breast, stomach, pancreas, prostate gland, bladder, bone, ovary, skin, kidney, hole, colon, intestines, stomach, rectum, esophagus, blood, brain and its coverture, spinal cord and its coverture, muscle, reticular tissue, suprarenal gland, Parathyroid, Tiroidina, uterus, testis, hypophysis, reproductive organ, liver, gall-bladder, Eye Ear Nose And Throat, tonsil, mouth and lymphoglandula and lymphsystem.

Other illness of available the inventive method treatment includes but not limited to: the tissue that virus, bacterium or parasite change, described tissue is selected from lung, breast, stomach, pancreas, prostate gland, bladder, bone, ovary, skin, kidney, hole, colon, intestines, stomach, rectum, esophagus, blood, brain and its coverture, spinal cord and its coverture, muscle, reticular tissue, suprarenal gland, Parathyroid, Tiroidina, uterus, testis, hypophysis, reproductive organ, liver, gall-bladder, Eye Ear Nose And Throat, tonsil, mouth and lymphoglandula and lymphsystem.

Illness to be treated also can be a tissue deformity or unusual, and described tissue is selected from lung, breast, stomach, pancreas, prostate gland, bladder, bone, ovary, skin, kidney, hole, colon, intestines, stomach, rectum, esophagus, blood, brain and its coverture, spinal cord and its coverture, muscle, reticular tissue, suprarenal gland, Parathyroid, Tiroidina, uterus, testis, hypophysis, reproductive organ, liver, gall-bladder, Eye Ear Nose And Throat, tonsil, mouth and lymphoglandula and lymphsystem.

Specifically be that illness to be treated can be the hypertrophy of tonsils, hyperplasia of prostate, psoriatic, eczema, tetter or hemorrhoid.Illness to be treated can be vascular disease such as atherosclerosis or arteriosclerosis, or vascular disease such as varix, artery or support is narrow or heavy narrow.Illness to be treated also can be that the beauty treatment of for example tissues such as skin, Eye Ear Nose And Throat, mouth, muscle, reticular tissue, hair or breast tissue is modified.

The therapeutic composition of S05A peptide belongs to the scope of the invention.This composition can comprise the S05A peptide with pharmaceutically acceptable carrier blended treatment significant quantity.Carrier substance can be a water for injection, preferably adds other material commonly used in the solution to be administered to Mammals.Usually, the S05A peptide that is used for the treatment of uses with composition forms, and composition comprises that the peptide of purifying is in conjunction with accepting carrier, vehicle or thinner on one or more physiology.Neutral buffered saline or with serum albumin blended salt solution be the example of suitable carrier.Preferably, product is made lyophilized products with appropriate excipients (as sucrose).Can comprise other standard vector, thinner and vehicle if desired.Composition comprises the suitable damping fluid of the known pH value of the those skilled in the art in this field scope, comprises the Tris damping fluid of about pH7.0-8.5 or the acetate buffer of about pH4.0-5.5, and damping fluid can further comprise sorbyl alcohol or its suitable surrogate.

Adopt coupling or connection or be incorporated into antibody, antibody fragment, antibody molecule or, have the S05A peptide of the molecule of high-affinity to come target unwanted cells element also to belong to the scope of the invention as the enzyme of cell receptor, signal peptide or overexpression to the specific tumour mark.Antibody, antibody fragment, antibody molecule or the molecule that specific tumour is marked with high-affinity is used for described peptide conjugate target specific cells or tissue.For example, there is the tumour of special surface antigen or antigen expressed can be killed by this peptide by antibody, antibody fragment, antibody sample binding molecule target and tumour cell.The advantage of the method for this use antibody target is the efficient that reduces dosage, improves combination and the possibility of being taken in by target cell, raising target and treatment metastatic tumo(u)r and microscopic size tumour.

The present invention also comprise adopt coupling or connection be incorporated into protein or the S05A peptide of other molecule forming composition, by tumour-specificity or site-specific enzymes or proteolytic enzyme or the antibody coupling matter by target tumor or other unwanted cells tumour or other unwanted cells position or near after the cutting can the position of tumour or other unwanted cells or near this peptide of release.

The present invention also comprise use coupling or connection be incorporated into protein or the S05A peptide of other molecule to form composition, be exposed to light (as laser therapy or other photodynamics or light activated therapy), other form electromagnetic radiation at tissue to be treated, after for example infrared radiation, ultraviolet radiation, x ray or gamma-rays radiation, local heating, α or β radiation, ultrasonic wave ray or other local energy source, composition discharges some bioactive fragments of this peptide or this peptide.

The S05A peptide can be separately, use together or in conjunction with the other medicines composition, as be suitable for cytokine, somatomedin, microbiotic, cell death inducer, anti-inflammatory and/or the chemotherapeutics of treatment indication.

The present invention also comprises use dendrimer, soccerballene and other synthetic molecules, polymkeric substance and macromolecular S05A composition, wherein said peptide and/or its corresponding dna molecular are by itself or with other kind quasi-molecule such as tumour-specific markers, with above-mentioned molecule, polymkeric substance or macromole coupling, combine or be contained in wherein.For example, U.S. Patent number 5,714,166 " the dendrimer conjugates of biological activity and/or target " (Bioactive and/or Targeted Dendrimer Conjugates) provide preparation and have used the method for tree-shaped polymer conjugates, and this conjugate comprises at least one dendrimer and leads target thing and at least a biologically active agent of link coupled with it.United States Patent (USP) 5,714,166 disclosed contents are totally included this paper in for your guidance.

The present invention also comprises the therapeutic composition of S05A and/or gene and drug carrier system, described carrier such as liplid emulsions, micellar copolymerization thing, polymer microballoon, electroactive polymer, hydrogel and liposome.

Use is transferred to the S05A that do not need cell or genes involved or gene coordinator and is also belonged to invention scope.The overexpression peptide can be used for the necrocytosis in the induced tumor in the tumour, thereby reduces tumor cell group.Gene or gene coordinator shift S05A with treatment unwanted cells element, thus estimate its advantage be required dosage still less, be passed on the cell offspring of target cell element and make the lower and overall treatment of therapeutic frequency still less.The present invention also comprises coding is contained the transgenosis of fusion rotein of S05A to unwanted cells or its adjacent cells, behind expressing gene and generation and/or the secretion fusion rotein, fusion rotein by natural enzyme or proteolytic enzyme or prodrug cutting with discharge this peptide to unwanted cells or near.

Use recombinant peptide-antibody coupling matter of clone; Recombinant peptide-antibody fragment conjugate of clone; Recombinant peptide-antibody sample protein conjugate of clone also belongs to invention scope.Except making and the advantage of the binding molecule that standardized production is cloned, clone's S05A peptide is the above-mentioned target advantage of this molecular combinations in conjunction with the advantage of target conjugate (as antibody, antibody fragment, antibody molecule or cancer specific receptors or other tumor marker are had the molecule of high-affinity).

The present invention also comprise adopt S05A peptide or gene or gene Equivalent therapeutic composition as bag by the composition of medical treatment device such as support, to eliminate, to suppress or prevention undesirable cell propagation or accumulation.

Orally administered solid formulation shape includes but not limited to capsule, tablet, pill, powder and particle.In this solid dosage form, active compound mixes with at least a following material: (a) one or more inert excipients (or carrier), as Trisodium Citrate or Lin Suanergai; (b) weighting agent or swelling agent are as starch, lactose, sucrose, glucose, N.F,USP MANNITOL and silicic acid; (c) tackiness agent is as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and Sudan Gum-arabic; (d) wetting Agent for Printing Inks is as glycerine; (e) disintegrating agent is as agar-agar, lime carbonate, potato or tapioca (flour), Lalgine, some composition silicate and yellow soda ash; (f) solution retarder is as paraffin; (g) absorb accelerator, as quaternary ammonium compound; (h) wetting agent is as the pure and mild Zerol of acetyl; (i) absorption agent is as kaolin and wilkinite; (j) slipping agent is as talcum powder, calcium stearate, Magnesium Stearate, solid polyethylene glycol, Sodium Lauryl Sulphate BP/USP and their mixture.With regard to capsule, tablet, pill, agent shape also can comprise buffer reagent.

Orally administered liquid agent shape comprises pharmaceutically acceptable emulsion, solution, suspension, syrup and elixir.Except active compound, liquid agent shape can comprise this area inert diluent such as water or other solvent, solubilizing agent and emulsifying agent commonly used.The emulsifying agent example is ethanol, Virahol, ethyl-carbonate, ethyl acetate, benzylalcohol, peruscabin, propylene glycol, 1,3-butyleneglycol, dimethyl formamide, oily as oleum gossypii seminis, peanut oil, Fructus Maydis oil, sweet oil, Viscotrol C and sesame oil, the fatty acid ester of glycerine, tetrahydrofurfuryl alcohol, polyoxyethylene glycol, sorbitanic or the mixture of these materials etc.

Except this inert diluent, composition also can comprise adjuvant, as wetting agent, emulsifying agent and suspension agent, sweeting agent, seasonings and flavouring agent.

With regard to concrete composition and application process, the actual dose level that can change activeconstituents in the invention composition is to obtain effectively to obtain the S05A peptide amount of required therapeutic response.Therefore select dosage level to depend on the time length and the other factors of required result of treatment, route of administration, required treatment.

Can give the significant quantity that Mammals comprises the people according to body surface area.Animal and human's the dosage mutual relationship that is used for different sizes, kind is (according to the M of mg/ body surface ²) be described in E.J.Freireich etc., CancerChemother.Rep., 50 (4): 219 (1966).Body surface area can roughly be determined (referring to for example " science table " (Scientific Tables), gigue pharmaceutical companies (Geigy Pharmaceuticals), A Desilei (Ardsley), New York 537-538 page or leaf (1970)) from individuality height and weight.

Total dose every day that is administered to host's S05A peptide can be a dosage single or that separate.Dosage unit compositions comprises the part of the consumption that constitutes dosage every day.Yet will be appreciated that, any concrete patient's given dose level depends on multiple factor, comprise body weight, holistic health, sex, diet, time of application and approach, drug administration effectiveness, absorption and excretory ratio, with the seriousness of the concrete illness of the combination of other medicines and treatment.

The method of using the S05A peptide combinations according to the present invention includes but not limited in intramuscular, per os, intravenously, intraperitoneal, the brain in (in the essence), Intraventricular, the tumour, in the intralesional, intradermal, sheath, in the nose, intraocular, intra-arterial, part, transdermal, by aerosol, infusion, inject, administered compounds such as transplantation device, slow-released system.

The method that another kind is used invention S05A peptide is through skin or transdermal route.An example of this embodiment is to use paster.Specifically be, can in the mixture of for example methyl-sulphoxide (DMSO) or DMSO and oleum gossypii seminis, the delicate suspensions with peptide prepare paster, make it to contact the mammal skin that carries tumour and away from the interior tumor-localizing of skin capsule (pouch) position.Other medium or its mixture that contain other solvent and solid support also can work equally.Paster can comprise the peptide compounds of solution or form of suspension.Paster can be applied to patient skin then, for example its be inserted by pin, clip or other clamping device skin is folding and be clamped together and in the patient skin capsule that forms.Should guarantee capsule and skin Continuous Contact and not disturbed by Mammals.Except using the skin capsule, can use any can guarantee paster firmly placed and with the device of skin contact.For example, available adhesion bandage is clamped in paster on the skin.

The S05A peptide can be used in sustained release preparation or goods.The suitable example of slowly-releasing goods comprises the semipermeable polymers matrix of formed article form, as film or microcapsule.Sustained-release matrix comprises polyester, hydrogel, the polylactide (U.S. 3,773,919, EP58,481), the multipolymer (Sidman etc. of L-L-glutamic acid and γ ethyl-L-glutaminate, Biopolymers, 22:547-556), poly-(2-hydroxyethyl-methacrylic ester) (Langer etc., J.Biomed.Mater.Res., 15:167-277 and Langer etc., Chem.Tech., 12:98-105), ethylene vinyl acetate (Langer etc., the same) or poly--D (-)-3-hydroxybutyric acid (EP133,988).Slow releasing composition also can comprise liposome, can be prepared by any certain methods known in the art (as Eppstein etc., Proc.Natl.Acad.Sci.USA, 82:3688-3692; EP 36,676; EP 88,046; EP 143,949).

The method that another kind is used S05A peptide of the present invention is to make the S05A peptide directly or indirectly be infused into tumour to be treated or other tissue.An example of this embodiment is that the direct injection peptide is to tumour or other tissue to be treated.Treatment can be by single injection, for some time multiple injection or a series of injections in during several hours, several days or some months form, monitor disappearing of tumour to be treated or other tissue or destroy by the method for examination of living tissue, imaging or other monitoring tissue growth.Be injected into tumour to be treated or other tissue can pass through patchhole, arrive intravital tumour or tissue as the device in nose, mouth, ear, vagina, rectum or the urethra or through otch, and can be combined into picture or optical system such as ultrasonic wave or fibrescope and carry out to determine the proper site of injection.Another example of this embodiment is to use the device that can make S05A peptide constant infusion tissue.

The another kind of method of using S05A peptide of the present invention is in conjunction with operation or with the physics excision, divests or kill or destroy tumour or need or wish to remove or destroy other tissue or the used similar process of cell element, S05A peptide wherein of the present invention is administered to around the immediate area in the zone of removing tumour or tissue, thereby destruction or prevention are not removed by this process or the growth of any tumour cell of destructive or other cell element.

The another kind of method of using S05A peptide of the present invention is that certain device is transplanted in tumour to be treated or other tissue.An example of this embodiment is that the thin slice that contains peptide is transplanted in tumour to be treated or other tissue.Thin slice passing in time is discharged into the peptide of therapeutic dose in the tissue.In addition, can be transplanted in the involved area and topical composition by film, sponge or other suitable substance that will absorb the S05A peptide.If the use transplantation device can be transplanted to this device any suitable tissue or organ, sending peptide can be directly by device, through injecting or adopting continuous infusion through continuous administration or through conduit.

Another kind of application process is the cell that the S05A peptide-encoding gene of one or more copies is imported target, if necessary, induces the gene of multiple copied to begin to produce in the born of the same parents peptide.But the mode of a kind of applying gene treatment is to use S05A peptide-encoding gene (synthetic DNA of genomic dna, cDNA and/or this peptide of encoding (or its fragment, variant, homologue or derivative)), and it can be operatively connected in composing type or inducible promoters to form " gene therapy DNA construction ".If promotor has activity in the cell or tissue type that construction inserts, it can with endogenous peptide-encoding gene homology or allos.If desired, other composition of gene therapy DNA construction can randomly comprise the dna molecular that the is designed for site-specific integration endogenous flanking sequence of homologous recombination (as be used for), tissue-specific promoter, one or more enhansers or one or more silencer can provide the dna molecular better than the selectivity advantage of parental cell, be used for the dna molecular of mark with identification of transformed cell, negative selective system, cell-specific wedding agent (for example being used for cell-targeting) the cell-specific internalization factor, the factor that strengthens the transcription factor of vector expression and carrier is produced.

In the body or the ex vivo delivered gene include, but is not limited to direct injection naked DNA, ballistic processes (ballistic method), liposome-mediated transfer, receptor-mediated transfer (part-dna complex), electroporation and calcium phosphate precipitation to the method in the cell or tissue, referring to U.S. Patent number 4,970,154, WO 96/40958, U.S. Patent number 5,679,559, U.S. Patent number 5,676,954 and U.S. Patent number 5,593,875, it is for reference that content is separately all included this paper in.Also comprise the employing virus vector,, adopt the DNA-protein conjugate and adopt liposome as retrovirus, adenovirus, adeno-associated virus, poxvirus, slow virus, papilloma virus or hsv.Report the application of gene therapy vector, seen United States Patent (USP) 5,672,344; 5,399,346; 5,631,236 and 5,635,399.These patents content whole is separately introduced for your guidance.

Can S05A peptide-encoding gene be delivered in some patient's cell by transplanting, it is genetically engineered to express and secretion S05A peptide or its fragment, variant, homologue or derivative that cell uses methods described herein to exsomatize.This cell can be animal or human's cell, can obtain from patient autologous tissue or from people or another inhuman source.Randomly, cell can be immortalization or stem cell.Yet for reducing the possibility of immunne response, preferably with the cell packing to prevent the infiltration of surrounding tissue.The packing material generally is biocompatible semipermeability polymerization closure or film, and they can make protein product discharge but prevent patient's immunity system or destroy cell from other adverse factor of surrounding tissue.The method that is used for the film cystocyte is that the technician is familiar with, and can prepare the cell of packing and is transplanted among the patient and does not need the over-drastic experiment.Referring to for example U.S. Patent number 4,892,538; 5,001,472 and 5,106,627, it is for reference that the content that they are announced is all included this paper in.The system description of packing viable cell is in PCT WO 91/10425.It is well known by persons skilled in the art making multiple other technology lasting or the controlled delivery mode, and as liposome vectors, biological erodible particle or pearl, these technical descriptions are in for example U.S. Patent number 5,653,975, and it is for reference that its content is all included this paper in.The cell portable that is with or without packing is in patient's suitable bodily tissue or organ.

It is to give one or more peptides of the present invention that treatment need remove or destroy in the special preferred implementation of method of illness of cell, and described needs are removed or the destructive cell here is a Lymphoid tissue.Other preferred implementations comprise that treatment need remove or destroy the illness of cell, and as any illness that is selected from down group alone or in combination: the beauty treatment of the hypertrophy of tonsils, hyperplasia of prostate, vascular disease (atherosclerosis or arteriosclerosis), hemorrhoid, varix, psoriatic, eczema, tetter and tissue is modified.Other medicable illnesss comprise: artery or narrow, the restenosis, sealing or the obstruction that are placed on or implant endarterial support.Medicable in a preferred embodiment suitable tissue comprises: skin, Eye Ear Nose And Throat, mouth, muscle, reticular tissue, hair or breast.

Other the preferred illnesss that can treat according to the present invention comprise those that are selected from down group: inflammatory diseases, autoimmune disease, metabolic trouble, genetic diseases, trauma or physiological damage, deficiency disease, transmissible disease, amyloid disease, fibrosis disease, thesaurismosis, congenital abnormality, enzymatic defect disease, poisoning, drunk, 20th-century disease, Radiation sickness, endocrinopathy, degenerative disease and functional disease.Other preferred embodiment in, described peptide is coupled to, is connected in or be incorporated into a kind of molecule, described molecule is selected from antibody, antibody fragment and antibody sample binding molecule, and wherein said molecule is higher than avidity in conjunction with other cell in conjunction with the avidity of tumour or other target.In other embodiments, described peptide is an a kind of part of recombinant molecule of new clone, described recombinant molecule is made of this peptide and certain molecule, described molecule is selected from antibody, antibody fragment and antibody sample binding molecule, and wherein said molecule is higher than avidity in conjunction with other cell in conjunction with the avidity of tumour or other target.

Provide following example to illustrate the present invention.Yet be understood that and the invention is not restricted to actual conditions described in these examples and details.In specification sheets, comprise that any of United States Patent (USP) and the available document of all public include in for your guidance specially.

Specifically, the present invention is particularly including pending U.S. Patent Application sequence number 10/092,934: the embodiment that lists in " with the method for using neural thread protein treatment tumour and associated conditions " for your guidance, this part application shows that complete AD7c-albumen is normal rodent muscle tissue in external glioma and neuroblastoma culture and in the body, the tumour in subcutaneous connective tissue and corium and multiple different people and inhuman source (comprises the mammary cancer in the rodent model, skin carcinoma and papilloma, colorectal carcinoma, cerebral glioma and other tumour) in cause the potent agent of necrocytosis.The present invention also particularly including pending U.S. Patent Application sequence number 10/153,334, is entitled as " effectively treat tumour and need remove or destroy the peptide of the illness of cell with other "; Sequence number 10/198,069 is entitled as " effectively treat tumour and need remove or destroy the peptide of the illness of cell with other "; Sequence number 10/198,070 is entitled as " effectively treat tumour and need remove or destroy the peptide of the illness of cell with other "; Sequence number 10/294,891 is entitled as " effectively treat tumour and need remove or destroy the peptide of the illness of cell with other "; And sequence number 10/920,313, be entitled as the embodiment that lists in " effectively treat tumour need remove or destroy the peptide of the illness of cell with other " for your guidance, they disclose wherein said some peptide separately is the potent agent that causes necrocytosis in normal in vivo rodent muscle tissue, subcutaneous connective tissue, corium and other tissue.

Embodiment 1

The purpose of this example is to determine the effect of S05A peptide to injection site tissue.

Following S05A peptide adopts standard method synthetic:

S05A-2(SEQ ID NO.2)IDQQVLSRI(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile)；

S05A-3(SEQ ID NO.3)KLEIKRCL(Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu)；

S05A-4(SEQ ID NO.4)VLSRIK(Val-Leu-Ser-Arg-Ile-Lys)；

S05A-5(SEQ ID NO.5)RIKLEIK(Arg-Ile-Lys-Leu-Glu-Ile-Lys)；

S05A-6(SEQ ID NO.6)VLSRIKLEIKRCL(Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu)；

S05A-7(SEQ ID NO.7)IDQQVLSRIKLEI(Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-GIu-IIe)。

Behind the prostate gland visual operation, give one of above-mentioned S05A peptide with etherization male Sprague-Dawley rat (300 gram weight scope) and by infusion in the prostate gland, only contrast injecting normal saline or do not inject.Injection by 300 μ l S05A peptides (being made into 1mg/ml) with PBS pH 7.4 (1.0mg/kg) (n=36) constitute.The back painless rat that kills of 24 hours (n=12), 72 hours (n=12) or 7 days (n=12).Extract prostate gland, in 10% buffered formalin, fix 24 hours, paraffin embedding, section, and use H﹠amp; E dyeing.The complete prostate gland of each animal is all done embedding and section.Check and measure all stained with Histological method.Each prostate gland is checked 2 tissue slicies at least, detects two square section diameters (D) (each prostate gland is measured altogether more than or equal to 8 times) that every tissue slice is 90 degree each other.According to V=4/3 π (D/2) ³With average cross-sectional diameter (D) the estimation volume of each group, and calculate the average-volume that each is organized.According to following criterion evaluation Histological change:

-do not have

+ current, minimum

++ current, medium

+++current, medium and disperse

++ ++ current, disperse and extensively

Conclusion: following table 4 has been listed the Histological change of observed necrocytosis.

Table 4

The S05A peptide	The Histological change of 24 hour cell death	The Histological change of 72 hour cell death
			S05A-2	++++	++++
S05A-3	+	+
			S05A-4	+	+
S05A-5	+	+
			S05A-6	+	+
S05A-7	++++	++++

The comparative study of before only injecting 1mg/mL PBS shows that inorganization changes or Histological change's minimum, comprise syringe needle (near) slight focal inflammation is arranged (referring to the embodiment in the following application: wait to examine U.S. Patent Application Serial Number 10/153,334: be entitled as " effectively treat tumour and other need remove or destroy the peptide of the illness of cell "; Sequence number 10/198,069 is entitled as " effectively treat tumour and need remove or destroy the peptide of the illness of cell with other "; Sequence number 10/198,070 is entitled as " effectively treat tumour and need remove or destroy the peptide of the illness of cell with other "; Sequence number 10/294,891 is entitled as " effectively treat tumour and need remove or destroy the peptide of the illness of cell with other "; And sequence number 10/920,313, be entitled as " effectively treat tumour and need remove or destroy the peptide of the illness of cell ", for your guidance) with other.

Following table 5 has been listed observed stereomutation.

Table 5

Compared with the control, the integral body of prostate volume reduces on average in the rat of expectation injection S05A peptide S05A-2 and S50A-7〉40%.The rat of handling with S50A-2 and S50A-7 demonstrates necrocytosis widely, necrosis, glandular epithelium loss and atrophy.Contrast demonstrates minimum change or does not change, and comprises that the kitchen range of the acute inflammation of injection site and syringe needle is little hemorrhage.

It will be understood by those skilled in the art that and to make multiple improvement and change to method and composition of the present invention and do not deviate from the spirit or scope of invention.

Claims (19)

1. isolating peptide, described peptide is made of the fragment of the peptide that aminoacid sequence shown in the SEQ ID NO.1 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu) constitutes.

2. peptide as claimed in claim 1 is characterized in that, described peptide is selected from down group:

A) peptide that constitutes by aminoacid sequence shown in the SEQ ID NO.2 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile);

B) peptide that constitutes by aminoacid sequence shown in the SEQ ID NO.3 (Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu);

C) peptide that constitutes by aminoacid sequence shown in the SEQ ID NO.4 (Val-Leu-Ser-Arg-Ile-Lys);

D) peptide that constitutes by aminoacid sequence shown in the SEQ ID NO.5 (Arg-Ile-Lys-Leu-Glu-Ile-Lys);

E) peptide that constitutes by aminoacid sequence shown in the SEQ ID NO.6 (Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu); With

F) peptide that constitutes by aminoacid sequence shown in the SEQ ID NO.7 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile).

3. composition, described composition comprises at least a peptide as claimed in claim 1 and carrier.

4. the stand-in of a peptide as claimed in claim 1.

5. isolating peptide, described peptide comprise at least two fragments of the peptide that is made of aminoacid sequence shown in the SEQ ID NO.1 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu).

6. isolating peptide, described peptide comprise segmental at least two repetitions of the peptide that is made of aminoacid sequence shown in the SEQ ID NO.1 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu).

7. isolating peptide, according to the segmental aminoacid sequence of the peptide that is made of aminoacid sequence shown in the SEQ ID NO.1 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu), the aminoacid sequence that this isolating peptide comprises is anti--D order.

8. isolating peptide, described peptide comprise the fragment of the peptide that is made of aminoacid sequence shown in the SEQ ID NO.1 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu) that merges with antibody, antibody fragment or antibody molecule.

9. isolating peptide, described peptide comprises the fragment of the peptide that is made of aminoacid sequence shown in the SEQ ID NO.1 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu) and at least 1 to 25 side joints are at the additional amino acid of this peptide 3 ' or 5 ' end at the most, and wherein said isolating peptide does not comprise the peptide by the formation of aminoacid sequence shown in the SEQ ID NO.1 (Ile-Asp-Gln-Gln-Val-Leu-Ser-Arg-Ile-Lys-Leu-Glu-Ile-Lys-Arg-Cys-Leu).

10. a nucleic acid, the described peptide of described nucleic acid encoding and claim 1 and the corresponding aminoacid sequence of homologue, fragment and variant thereof.

11. a composition, described composition comprise one or more nucleic acid nucleic acid as claimed in claim 10 and pharmaceutically acceptable carrier.

12. the method for the mammiferous illness of treatment, described illness requires to remove or destroy cell, and described method comprises the peptide as claimed in claim 1 that gives described Mammals treatment significant quantity.

13. method as claimed in claim 12 is characterized in that, described peptide is by being selected from down the method afford of group: in per os, subcutaneous, intradermal, the nose, in the intravenously, intramuscular, sheath, in the nose, in the tumour, local and transdermal.

14. method as claimed in claim 12, it is characterized in that, be selected from before down the methods of treatment of group is treated this Mammals, during or afterwards described Mammals is implemented described method: excision, transplanting, grafting, chemotherapy, immunotherapy, vaccine inoculation, heat or electricity melt, psychrotherapy, laser therapy, phototherapy, gene therapy and radiotherapy.

15. method as claimed in claim 12, it is characterized in that described illness is the optimum or malignant tumour that is selected from down the tissue of group: lung, breast, stomach, pancreas, prostate gland, bladder, bone, ovary, skin, kidney, hole, colon, intestines, stomach, rectum, esophagus, the heart, spleen, sialisterium, blood, brain and its coverture, spinal cord and its coverture, muscle, reticular tissue, suprarenal gland, Parathyroid, Tiroidina, uterus, testis, hypophysis, reproductive organ, liver, gall-bladder, Eye Ear Nose And Throat, tonsil, mouth and lymphoglandula and lymphsystem.

16. method as claimed in claim 12, it is characterized in that described illness is the hyperplasia that is selected from down the tissue of group, hypertrophy or hypertrophy: lung, breast, stomach, pancreas, prostate gland, bladder, bone, ovary, skin, kidney, hole, colon, intestines, stomach, rectum, esophagus, the heart, spleen, sialisterium, blood, brain and its coverture, spinal cord and its coverture, muscle, reticular tissue, suprarenal gland, Parathyroid, Tiroidina, the uterus, testis, hypophysis, reproductive organ, liver, gall-bladder, eye, ear, nose, larynx, tonsil, mouthful, and lymphoglandula and lymphsystem.

17. method as claimed in claim 12, it is characterized in that, described illness is to be subjected to virus, the tissue that bacterium or parasite change, described tissue is selected from: lung, breast, stomach, pancreas, prostate gland, bladder, bone, ovary, skin, kidney, hole, colon, intestines, stomach, rectum, esophagus, the heart, spleen, sialisterium, blood, brain and its coverture, spinal cord and its coverture, muscle, reticular tissue, suprarenal gland, Parathyroid, Tiroidina, the uterus, testis, hypophysis, reproductive organ, liver, gall-bladder, eye, ear, nose, larynx, tonsil, mouthful, and lymphoglandula and lymphsystem.

18. method as claimed in claim 12, it is characterized in that described illness is the deformity that is selected from down the tissue of group: lung, breast, stomach, pancreas, prostate gland, bladder, bone, ovary, skin, kidney, hole, colon, intestines, stomach, rectum, esophagus, the heart, spleen, sialisterium, blood, brain and its coverture, spinal cord and its coverture, muscle, reticular tissue, suprarenal gland, Parathyroid, Tiroidina, uterus, testis, hypophysis, reproductive organ, liver, gall-bladder, Eye Ear Nose And Throat, tonsil, mouth and lymphoglandula and lymphsystem.

19. comprising with the peptide according to claim 1 for the treatment of significant quantity at least, a prevention or suppress narrow, the sealing of support or the method for stopping up, described method be coated with this support.